The present invention relates to transgenic animals, compositions and methods relating to the characterization of gene function.
Normal growth and differentiation of all organisms is dependent on cells responding correctly to a variety of internal and external signals. Many of these signals produce their effects by ultimately changing the transcription of specific genes. One well-studied group of proteins that mediate a cell""s response to a variety of signals is the family of transcription factors known as nuclear receptors. Members of this group include receptors for steroid hormones, vitamin D, ecdysone, cis and trans retinoic acid, thyroid hormone, fatty acids (and other peroxisomal proliferators), as well as so-called orphan receptors, proteins that are structurally similar to other members of this group, but for which no ligands are known. Orphan receptors may be indicative of unknown signaling pathways in the cell or may be nuclear receptors that function without ligand activation. There are indications that the activation of transcription by some of these orphan receptors may occur in the absence of an exogenous ligand and/or through signal transduction pathways originating from the cell surface.
Steroid hormones affect the growth and function of specific cells by binding to intracellular receptors (SR) and forming SR-hormone complexes. SR-hormone complexes then interact with a hormone response element (HRE) in the control region of specific genes and alter specific gene expression. cDNAs for many SRs have been isolated and characterized, making it possible to deduce the amino acid sequences of various steroid/thyroid/retinoic acid receptors and related members of the super family of nuclear receptors (Evans et al., Science, 240:889-895 (1988); Liao et al., J. Steroid Biochem., 34:(1-6) 41-51 (1989); Forman et al., New Biol., 587-594 (1990)).
The complete coding sequences for human (AF148128) and murine (AF148129; SEQ ID NO:21) retina-specific nuclear receptors were determined and published by Chen et al. (Proc. Natl. Acad. Sci. U.S.A. 96(26), 15149-15154 (1999)). According to Chen et al., human RNR is a splice variant of PNR. Northern blot and reverse transcription-PCR analyses of human mRNA samples demonstrated that RNR is expressed exclusively in the retina, with transcripts of approximately 7.5 kb, approximately 3.0 kb, and approximately 2.3 kb by Northern blot analysis. In situ hybridization with multiple probes on both primate and mouse eye sections demonstrated that RNR is expressed in the retinal pigment epithelium and in Muller glial cells. By using the Gal4 chimeric receptor/reporter cotransfection system, the ligand binding domain of RNR was found to repress transcriptional activity in the absence of exogenous ligand. Gel mobility shift assays revealed that RNR can interact with the promoter of the cellular retinaldehyde binding protein gene in the presence of retinoic acid receptor (RAR) and/or retinoid X receptor (RXR).
Given the importance of retinoic acid receptors in the regulation of gene expression, a clear need exists for further characterization of these receptors which can play a role in preventing, ameliorating or correcting dysfunctions or diseases.
The present invention generally relates to transgenic animals, as well as to compositions and methods relating to the characterization of gene function. More specifically, the present invention relates to nucleic acid sequences encoding a retina-specific nuclear receptor and in the in vivo characterization of genes encoding a retina-specific nuclear receptor.
The present invention provides transgenic cells comprising a disruption in retina-specific nuclear receptor gene. Preferably, the transgenic cells of the present invention are stem cells and more preferably, embryonic stem (ES) cells, and most preferably, murine ES cells. Preferably, the target gene""s coding sequence (i.e., exons) comprises SEQ ID NO:21. According to one embodiment, the transgenic cells are produced by introducing a targeting construct into a stem cell to produce a homologous recombinant, resulting in a disruption of the target sequence encoding a retina-specific nuclear receptor. In another embodiment, the transgenic cells are derived from the transgenic animals described below.
The present invention also provides a targeting construct and methods of producing the targeting construct that when introduced into stem cells produces a homologous recombinant generating transgenic cells comprising a disruption in a retina-specific nuclear receptor. In one embodiment, the targeting construct of the present invention comprises first and second polynucleotide sequences that are homologous to the target sequence. The targeting construct also comprises a polynucleotide sequence that encodes a positive selection marker that is preferably positioned between the two different homologous polynucleotide sequences in the construct.
The present invention further provides non-human transgenic animals comprising a disruption in a retina-specific nuclear receptor gene and methods of producing such transgenic animals. The transgenic animals of the present invention include transgenic animals that are heterozygous and homozygous for a mutation in the gene that naturally encodes and expresses a functional retina-specific nuclear receptor gene. In one aspect, the transgenic animals of the present invention are defective in the function of the retina-specific nuclear receptor gene. The present invention also encompasses cells and cell lines derived from the transgenic animals of the present invention.
The transgenic animals of the present invention further comprise a phenotype associated with having a defect or disruption in a retina-specific nuclear receptor gene.
The present invention also provides a method of identifying agents capable of affecting a phenotype of a transgenic animal. According to this method, a putative agent is administered to a transgenic animal. The response of the transgenic animal to the putative agent is then measured and compared to the response of a xe2x80x9cnormalxe2x80x9d or wild type mouse, or alternatively compared to a transgenic animal control (without agent administration). The invention further provides agents identified according to such methods.
The present invention further provides a method of identifying agents having an effect on retina-specific nuclear receptor gene expression or function. The method includes administering an effective amount of the agent to a transgenic animal, preferably a mouse, having a disruption in a retina-specific nuclear receptor gene. The method includes measuring a response of the transgenic animal, for example, to the agent, and comparing the response of the transgenic animal to a control mouse. The response of the transgenic animal as compared to the control mouse may serve as an indication of the specificity or activity of the agent. Compounds that may have an effect on retina-specific nuclear receptor gene expression or function may also be screened against cells in cell-based assays, for example, to identify such compounds.
The present invention also provides methods of identifying agents useful as therapeutic agents for treating conditions associated with a disruption in a retina-specific nuclear receptor gene. In a preferred embodiment, conditions include those associated with the phenotypes of the mice of the present invention. In accordance with this method, the present invention provides animal models useful in identifying compounds that are able to affect a phenotype, such as a physiological or behavioral phenotype associated with a disruption of a retina-specific nuclear receptor gene. The method involves, for example, administering a putative agent to a transgenic animal. The response of the transgenic animal to the putative agent is then measured and compared to the response of a xe2x80x9cnormalxe2x80x9d or wild-type mouse, or alternatively compared to a transgenic animal control (without agent administration). The invention further provides agents identified according to such methods.
The invention also provides cell lines comprising nucleic acid sequences encoding a retina-specific nuclear receptor. Such cell lines may be capable of expressing such sequences by virtue of operable linkage to a promoter functional in the cell line. Preferably, expression of the sequence encoding a retina-specific nuclear receptor is under the control of an inducible promoter. Also provided are methods of identifying agents that interact with retina-specific nuclear receptor, comprising the steps of contacting a retina-specific nuclear receptor with an agent and detecting an agent/retina-specific nuclear receptor complex. Such complexes can be detected by, for example, measuring expression of an operably linked detectable marker.
The invention further provides methods of treating diseases or conditions associated with a disruption in a gene encoding a retina-specific nuclear receptor, and more particularly, to a disruption in the expression or function of a retina-specific nuclear receptor. In a preferred embodiment, methods of the present invention involve treating diseases or conditions associated with a disruption in retina-specific nuclear receptor expression or function, including administering to a subject in need, a therapeutic agent which effects retina-specific nuclear receptor expression or function. In accordance with this embodiment, the method comprises administration of a therapeutically effective amount of a natural, synthetic, semi-synthetic, or recombinant retina-specific nuclear receptor or fragment thereof as well as natural, synthetic, semi-synthetic or recombinant analogs.
The present invention further provides methods of treating diseases or conditions associated with disrupted retina-specific nuclear receptor expression or function, wherein the methods comprise detecting and replacing through gene therapy mutated retina-specific nuclear receptor genes.
As used herein, xe2x80x9cgenexe2x80x9d refers to (a) a gene containing at least one of the DNA sequences disclosed herein; (b) any DNA sequence that encodes the amino acid sequence encoded by the DNA sequences disclosed herein and/or; (c) any DNA sequence that hybridizes to the complement of the coding sequences disclosed herein. Preferably, the term includes coding as well as noncoding regions, and preferably includes all sequences necessary for normal gene expression including promoters, enhancers and other regulatory sequences.
As used herein, xe2x80x9cgene targetingxe2x80x9d is a type of homologous recombination that occurs when a fragment of genomic DNA is introduced into a mammalian cell and that fragment locates and recombines with endogenous homologous sequences.
xe2x80x9cDisruptionxe2x80x9d of a target gene occurs when a fragment of genomic DNA locates and recombines with an endogenous homologous sequence such that production of the normal wild type gene product is inhibited or functionally disrupted, resulting in, for example, partial or complete loss of expression of a protein encoded by a target gene. Non-limiting examples of disruption include insertion, missense, frameshift and deletion mutations. Gene targeting can also alter a promoter, enhancer, or splice site of a target gene to cause disruption, and can also involve replacement of a promoter with an exogenous promoter such as an inducible promoter described below.
As used herein, a xe2x80x9ctransgenic animalxe2x80x9d is an animal that contains within its genome a specific gene that has been disrupted or inactivated completely or partially by the method of gene targeting. The transgenic animal includes both the heterozygote animal (i.e., one defective allele and one wild-type allele) and the homozygous animal (i.e., two defective alleles).
The terms xe2x80x9cpolynucleotidexe2x80x9d and xe2x80x9cnucleic acid moleculexe2x80x9d are used interchangeably to refer to polymeric forms of nucleotides of any length. The polynucleotides may contain deoxyribonucleotides, ribonucleotides and/or their analogs. Nucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The term xe2x80x9cpolynucleotidexe2x80x9d includes single-, double-stranded and triple helical molecules.
xe2x80x9cOligonucleotidexe2x80x9d refers to polynucleotides of between 5 and about 100 nucleotides of single- or double-stranded DNA. Oligonucleotides are also known as oligomers or oligos and may be isolated from genes, or chemically synthesized by methods known in the art. A xe2x80x9cprimerxe2x80x9d refers to an oligonucleotide, usually single-stranded, that provides a 3xe2x80x2-hydroxyl end for the initiation of enzyme-mediated nucleic acid synthesis.
The following are non-limiting embodiments of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A nucleic acid molecule may also comprise modified nucleic acid molecules, such as methylated nucleic acid molecules and nucleic acid molecule analogs. Analogs of purines and pyrimidines are known in the art, and include, but are not limited to, aziridinycytosine, 4-acetylcytosine, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, pseudouracil, 5-pentylnyluracil and 2,6-diaminopurine. The use of uracil as a substitute for thymine in a deoxyribonucleic acid is also considered an analogous form of pyrimidine.
A xe2x80x9cfragmentxe2x80x9d of a polynucleotide is a polynucleotide comprised of at least 9 contiguous nucleotides, preferably at least 15 contiguous nucleotides and more preferably at least 45 nucleotides, of coding or non-coding sequences.
As used herein, xe2x80x9cbase pair,xe2x80x9d also designated xe2x80x9cbp,xe2x80x9d refers to the complementary nucleic acid molecules. In DNA there are four xe2x80x9ctypesxe2x80x9d of bases: the purine base adenine (A) is hydrogen bonded with the pyrimidine base thymine (T), and the purine base guanine (G) with the pyrimidine base cytosine (C). Each hydrogen bonded base pair set is also known as a Watson-Crick base-pair. A thousand base pairs is often called a kilobase pair, or kb. A xe2x80x9cbase pair mismatchxe2x80x9d refers to a location in a nucleic acid molecule in which the bases are not complementary Watson-Crick pairs. The phrase xe2x80x9cdoes not include at least one type of base at any positionxe2x80x9d refers to a nucleotide sequence which does not have one of the four bases at any position. For example, a sequence lacking one nucleotide (i.e., lacking one type of base) could be made up of A, G, T base pairs and contain no C residues.
As used herein, the term xe2x80x9cconstructxe2x80x9d refers to an artificially assembled DNA segment to be transferred into a target tissue, cell line or animal, including human. Typically, the construct will include the gene or a sequence of particular interest, a marker gene and appropriate control sequences. The term xe2x80x9cplasmidxe2x80x9d refers to an autonomous, self-replicating extrachromosomal DNA molecule. In a preferred embodiment, the plasmid construct of the present invention contains a positive selection marker positioned between two flanking regions of the gene of interest. Optionally, the construct can also contain a screening marker, for example, green fluorescent protein (GFP). If present, the screening marker is positioned outside of and some distance away from the flanking regions.
The term xe2x80x9cpolymerase chain reactionxe2x80x9d or xe2x80x9cPCRxe2x80x9d refers to a method of amplifying a DNA base sequence using a heat-stable polymerase such as Taq polymerase, and two oligonucleotide primers; one complementary to the (+)-strand at one end of the sequence to be amplified and the other complementary to the (xe2x88x92)-strand at the other end. Because the newly synthesized DNA strands can subsequently serve as additional templates for the same primer sequences, successive rounds of primer annealing, strand elongation, and dissociation produce exponential and highly specific amplification of the desired sequence. PCR also can be used to detect the existence of the defined sequence in a DNA sample. xe2x80x9cLong-rangexe2x80x9d refers to PCR conditions which allow amplification of large nucleotides stretches, for example, greater than 1 kb.
As used herein, the term xe2x80x9cpositive selection markerxe2x80x9d refers to a gene encoding a product that enables only the cells that carry the gene to survive and/or grow under certain conditions. For example, plant and animal cells that express the introduced neormycin resistance (Neor) gene are resistant to the compound G418. Cells that do not carry the Neor gene marker are killed by G418. Other positive selection markers will be known to those of skill in the art.
xe2x80x9cPositive-negative selectionxe2x80x9d refers to the process of selecting cells that carry a DNA insert integrated at a specific targeted location (positive selection) and also selecting against cells that carry a DNA insert integrated at a non-targeted chromosomal site (negative selection). Non-limiting examples of negative selection inserts include the gene encoding thymidine kinase (tk). Genes suitable for positive-negative selection are known in the art, see e.g., U.S. Pat. No. 5,464,764.
xe2x80x9cScreening markerxe2x80x9d or xe2x80x9creporter genexe2x80x9d refers to a gene that encodes a product that can readily be assayed. For example, reporter genes can be used to determine whether a particular DNA construct has been successfully introduced into a cell, organ or tissue. Non-limiting examples of screening markers include genes encoding for green fluorescent protein (GFP) or genes encoding for a modified fluorescent protein. xe2x80x9cNegative screening markerxe2x80x9d is not to be construed as negative selection marker; a negative selection marker typically kills cells that express it.
The term, xe2x80x9cvectorxe2x80x9d refers: to a DNA molecule that can carry inserted DNA and be perpetuated in a host cell. Vectors are also known as cloning vectors, cloning vehicles or vehicles. The term includes vectors that function primarily for insertion of a nucleic acid molecule into a cell, replication vectors that function primarily for the replication of nucleic acid, and expression vectors that function for transcription and/or translation of the DNA or RNA. Also included are vectors that provide more than one of the above functions. In a preferred embodiment, the vector contains sites useful in the methods described herein, for example, the vectors xe2x80x9cpDG2xe2x80x9d or xe2x80x9cpDG4xe2x80x9d as described herein.
A xe2x80x9chost cellxe2x80x9d includes an individual cell or cell culture which can be or has been a recipient for vector(s) or for incorporation of nucleic acid molecules and/or proteins. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent due to natural, accidental, or deliberate mutation. A host cell includes cells transfected with the constructs of the present invention.
The term xe2x80x9cgenomic libraryxe2x80x9d refers to a collection of clones made from a set of randomly generated overlapping DNA fragments representing the genome of an organism. A xe2x80x9ccDNA libraryxe2x80x9d (complementary DNA library) is a collection of mRNA molecules present in a cell, tissue, or organism, turned into cDNA molecules with the enzyme reverse transcriptase, then inserted into vectors (other DNA molecules which can continue to replicate after addition of foreign DNA). Exemplary vectors for libraries include bacteriophage (also known as xe2x80x9cphagexe2x80x9d), which are viruses that infect bacteria, for example lambda phage. The library can then be probed for the specific cDNA (and thus mRNA) of interest. In one embodiment, library systems which combine the high efficiency of a phage vector system with the convenience of a plasmid system (for example, ZAP system from Stratagene, La Jolla, Calif.) are used in the practice of the present invention.
The term xe2x80x9chomologous recombinationxe2x80x9d refers to the exchange of DNA fragments between two DNA molecules or chromatids at the site of homologous nucleotide sequences, i.e., those sequences preferably having at least about 70 percent sequence identity, typically at least to about 85 percent identity, and preferably at least about 90 percent identity. Homology can be determined using a xe2x80x9cBLASTNxe2x80x9d algorithm. It is understood that homologous sequences can accommodate insertions, deletions and substitutions in the nucleotide sequence. Thus, linear sequences of nucleotides can be essentially identical even if some of the nucleotide residues do not precisely correspond or align.
As used herein the term xe2x80x9cligation-independent cloningxe2x80x9d is used in the conventional sense to refer to incorporation of a DNA molecule into a vector or chromosome without the use of kinases or ligases. Ligation-independent cloning techniques are described, for instance, in Aslanidis and de Jong, Nucleic Acids Res., 18:6069-74 and U.S. patent application Ser. No. 07/847,298 (1991).
As used herein, the term xe2x80x9ctarget sequencexe2x80x9d (alternatively referred to as xe2x80x9ctarget gene sequencexe2x80x9d or xe2x80x9ctarget DNA sequencexe2x80x9d) refers to the nucleic acid molecule with any polynucleotide having a sequence in the general population that is not associated with any disease or discernible phenotype. It is noted that in the general population, wild-type genes may include multiple prevalent versions that contain alterations in sequence relative to each other and yet do not cause a discernible pathological effect. These variations are designated xe2x80x9cpolymorphismsxe2x80x9d or xe2x80x9callelic variations.xe2x80x9d
In a preferred embodiment, the target DNA sequence comprises a portion of a particular gene or genetic locus in the individual""s genomic DNA. The target DNA sequence encodes a retina-specific nuclear receptor. According to one embodiment, the target DNA comprises part of a particular gene or genetic locus in which the function of the gene product is not known, for example, a gene identified using a partial cDNA sequence such as an EST. The target retina-specific nuclear receptor gene comprises the coding sequence represented by SEQ ID NO:21 or a naturally occurring allelic variation or homologue of the target gene.
The term xe2x80x9cexonucleasexe2x80x9d refers to an enzyme that cleaves nucleotides sequentially from the free ends of a linear nucleic acid substrate. Exonucleases can be specific for double or single-stranded nucleotides and/or directionally specific, for instance, 3xe2x80x2-5xe2x80x2 and/or 5xe2x80x2-3xe2x80x2. Some exonucleases exhibit other enzymatic activities, for example, T4 DNA polymerase is both a polymerase and an active 3xe2x80x2-5xe2x80x2 exonuclease. Other exemplary exonucleases include exonuclease III which removes nucleotides one at a time from the 5xe2x80x2-end of duplex DNA which does not have a phosphorylated 3xe2x80x2-end, exonuclease VI which makes oligonucleotides by cleaving nucleotides off of both ends of single-stranded DNA, and exonuclease lambda which removes nucleotides from the 5xe2x80x2 end of duplex DNA which have 5xe2x80x2-phosphate groups attached to them.
The term xe2x80x9crecombinasexe2x80x9d encompasses enzymes that induce, mediate or facilitate recombination, and other nucleic acid modifying enzymes that cause, mediate or facilitate the rearrangement of a nucleic acid sequence, or the excision or insertion of a first nucleic acid sequence from or into a second nucleic acid sequence. The xe2x80x9ctarget sitexe2x80x9d of a recombinase is the nucleic acid sequence or region that is recognized (e.g., specifically binds to) and/or acted upon (excised, cut or induced to recombine) by the recombinase. As used herein, the expression xe2x80x9cenzyme-directed site-specific recombinationxe2x80x9d is intended to include the following three events:
1. deletion of a pre-selected DNA segment flanked by recombinase target sites;
2. inversion of the nucleotide sequence of a pre-selected DNA segment flanked by recombinase target sites; and
3. reciprocal exchange of DNA segments proximate to recombinase target sites located on different DNA molecules.